Accumulative Characteristics of Pesticide Residues in Organs of Bivalves ( Anodonta woodiana and Corbicula leana ) Under Natural Conditions

Arch. Environ. Contam. Toxicol. 40, 35– 47 (2001) DOI: 10.1007/s002440010146

A R C H I V E S O F

Environmental Contamination a n d Toxicology © 2001 Springer-Verlag New York Inc.

Accumulative Characteristics of Pesticide Residues in Organs of Bivalves (Anodonta woodiana and Corbicula leana) Under Natural Conditions S. Uno,1* H. Shiraishi,2 S. Hatakeyama,2 A. Otsuki,1 J. Koyama3 1 2 3

Tokyo University of Fisheries, 4-5-7, Kohan, Minato-ku, Tokyo, 108-8477, Japan National Institute for Environmental Studies, 16-2, Onogawa, Tsukuba, Ibaraki, 305-0053, Japan National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5, Maruishi, Ohno, Saeki, Hiroshima, 739-0452, Japan

Received: 9 December 1999/Accepted: 9 July 2000

Abstract. Accumulative characteristics of pesticide residues in the gill, midgut gland, gonad, and the remaining tissues of the bivalve mollusks Anodonta woodiana and Corbicula leana were examined during the rice planting seasons of 1992 and 1994. Although seven pesticides, except thiobencarb, were accumulated all at ppb levels in the midgut gland (liver) and gonad of both bivalves during their application period, thiobencarb was accumulated in C. leana at extremely high levels of 15.70 ␮g g⫺1 in 1992 and 12.45 ␮g g⫺1 in 1994 in the midgut gland and 15.80 ␮g g⫺1 in 1992 and 16.40 ␮g g⫺1 in 1994 in the gonad, respectively. These levels were about 100 times higher than those in A. woodiana. Thiobencarb and molinate in A. woodiana and chlornitrofen (CNP) and molinate in C. leana remained in the gonad and midgut gland longer than in the gill and remaining tissues, while thiobencarb in organs of C. leana remained at ppm levels until the end of the experiments. The present study suggests that these interspecies differences can be attributed to differences between the two species in their ability to eliminate pesticides.

Filter-feeding shellfish in Japan are widely distributed and have large populations in rivers. Invertebrates have lower metabolic rates of xenobiotics than do fish; consequently, shellfish are reported to have higher contaminant residue levels than fish. Several papers have reported that tissue concentrations of chemicals can serve as integrative indices of bioavailable contaminants in the aquatic environment (e.g., Esser 1986; Stegeman and Lech 1991; Rhodes et al. 1997). Pesticides are introduced into river waters by direct application, spray drift, and agricultural runoff. The use of highly lipophilic and persistent pesticides, such as organochlorine

*Present address: Environmental Division, National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5, Maruishi Ohno, Hiroshima, 739-0452; Japan Correspondence to: S. Uno

compounds has been restricted in advanced countries because the chemicals are biomagnified in aquatic animals through the food web. The most toxic have potential to damage fishery resources and affect the health of those animals and humans that consume contaminated fish and shellfish. However, many rivers and streams in the plains of Japan are still being contaminated by various pesticide residues, particularly during the rice planting season, resulting in degraded river ecosystems (Hatakeyama et al. 1991). The concentration and composition of pesticide residues in river waters change daily, and large seasonal variations in their concentrations there, ranging from undetectable to ppb levels, are caused by such factors as drainage from rice fields after application of pesticides, microbial decomposition in water, and their adsorption onto suspended matter. Thus, accumulation patterns of pesticide residues by aquatic animals in river ecosystems are complicated, and the bioconcentration (accumulation of chemicals in water through gill) process through respiration, rather than biomagnification (accumulation of chemicals in foods by feeding) through the food web, can be an important factor, leading to the disturbance of river ecosystems (Hatakeyama et al. 1991). The standard method for assessing lethal toxicity of chemicals to aquatic organisms, exposure of the organism to toxic chemicals, needs periodic observation of the effects. Since the results are usually expressed as 50% lethal concentration (LC50), we often miss early symptoms of the toxic effects within organs of the test organisms. However, it would be expected that there are systematic relationships among the concentrations of chemicals in water and bioconcentration factors, distribution to each organ, tissue concentration, and toxicity, and it is necessary that the assessing of influence for chemicals to organisms is taken account of the relation of these factors (Connell 1995). Several laboratory experiments on the accumulation of chemicals in organs by shellfish through respiration. Roberts (1975) reported that the major accumulation site of endosulfan in tissues of Chlamys opercularis and Mytilus edulis in a 2-week exposure was the digestive gland. Watanabe et al. (1985) reported that when M. edulis was exposed to molinate, thiobencarb, chlornitrofen (CNP), and chromethoxinyl for 9

36

S. Uno et al.

Table 1. Pesticides measured in bivalves and river water No.

RT (min) Common Namea

1 2 3 4 5 6 7 8 9 10 11 12 13 14

12.7 13.4 15.4 16.1 16.4 17.0 18.2 19.1 19.4 19.8 19.9 23.4 24.4 24.8

Molinate (H) BPMC (I) CAT (H) Diazinon (I) TPN (F) IBP (F) Simetryn (H) MEP (I) Malathion (I) Thiobencarb (H) MPP (I) Butachlor (H) Pretilachlor (H) Oxadiazon (H)

15 16 17

27.6 27.8 32.4

CNP (H) EDDP (F) Mefenacet (H)

Chemical Name S-ethyl hexahydro-1H-azepine-1-carbothioate o-sec-butylphenyl methylcarbamate 2-chloro-4,6-bis (ehylamino)-1,3,5-triazine diethyl 2-isopropyl-4-methyl-6-pyrimidinyl phosphorothioate tetrachloroisophthalonitrile S-benzyl diisopropyl phosphorothioate 2,4-bis (ethylamino)-6-methylthio-1,3,5-triazine dimethyl 4-nitoro-m-tolyl phosphorothioate S-1,2-bis (ethoxycarbonyl)ethyl dimethyl phosphorodithioate S-o-chlorobenzyl diethylthiocarbamate dimethyl 4-methylthio-m-tolyl phosphorothioate 2-chloro-2⬘,6⬘-diethyl-N-(butoxymethyl)-acetanilide 2-chloro-2⬘,6⬘-diethyl-N-(propoxymethyl)-acetanilide 5-tret-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazolin2-one p-nitrophenyl 2,4,6-trichlorophenyl ether O-ethyl diphenyl phosphorodithioate 2-benzothiazol-2-yloxy-N-methylacetanilide

Solubility (mg/L)

log Kow

BCF (Bivalve)b

BCF (Snail)c

1,000 610 400 40 0.6 1,000 185 14 145 30 55 20 50 0.7

3.21 2.78 2.18 3.81 2.90 3.21 2.54 3.30 2.36 3.40 4.09 4.50 4.08 4.80

68

2

152

9

1,800

40

259 39 700

22 210

13,900

500

438

72

0.25 5 4

4.70 2.66 3.23

a

(H) herbicide, (I) insecticide, (F) fungicide Corbicula leana (Uno et al. 1997) c Cipangopludina chinensis (Uno et al. 1997) b

days, the chemicals accumulated in the gonad and midgut gland. Rhodes et al. (1997) reported that when Mya arenaria was exposed to [3H]TCDD for 24 h, its concentrations were highest in gonad tissue. However, there has been little research on the accumulative characteristics in individual organs of chemicals in shellfish in the field, except for a study by Yamagishi and Akiyama (1981), reporting that in Tapes philippinarum, the concentration of CNP residues in viscera was five times higher than that in muscle in Tokyo Bay during May to September. It is important to clarify the accumulative characteristics of shellfish organs under natural conditions in order to predict early effects of contaminants that bioconcentrate in aquatic biota. In the previous paper (Uno et al. 1997), we estimated the bioconcentration factors (BCFs) for several pesticides in the bivalve Corbicula leana and the river snail Cipangopludina chinensis under natural conditions using a first-order compartment model; we found that all pesticides accumulated at high levels in the bivalves in spite of large changes in pesticide residues concentrations in river. Pesticides remained longer (4 weeks) in C. leana than in the river snails. The BCFs of CNP, thiobencarb, and oxadiazon in C. leana (13,900, 3,050, and 700, respectively) were higher than those of other pesticides. The present paper describes the accumulation characteristics of pesticide residues in organs of two bivalve species (Anodonta woodiana and C. leana), based on daily changes in concentrations of pesticide residues for about 3 months under natural conditions, and interspecific differences. A. woodiana is a large bivalve (maximum shell length, 15 cm); C. leana is relatively small (maximum shell length, 3 cm). A. woodiana lives in the bottom of lake and pond and bed of river and is oviparous. That glochidium larva clings to fish gill and grows to assimilation of the fish fluid. A. woodiana isn’t edible for human, but this bivalve is important for spawning bed of bitterling fish (the genus Paracheilognathus). On the

other hand, C. leana lives in the sandy bed of river and stream and is ovoviviparous. It is edible in Japan. Both species occupy higher positions in the food chain in the river ecosystem and have a similar respiratory system and feeding habit: they take in dissolved oxygen through an inhalant siphon and simultaneously feed on suspended particles by filtering the water through their gills (Barnes 1968; Shiino 1969).

Methods and Materials The Model Stream An artificial stream was constructed next to the Kokai River, which is flanked by paddies along most of its length (Uno et al. 1997). River water was continuously pumped into the artificial stream, which had a length of 4 m, a width of 40 cm, and a height of 40 cm. The current speed (12–18 cm s⫺1), the water level (16 cm), and the discharge rate (about 9.6 L s⫺1) were controlled by a gate valve. River sediments, which were collected from the streambed where C. leana were gathered, were redistributed on the bottom of the artificial stream to a depth of 10 cm.

Reagents Table 1 shows a list of 17 pesticides (9 herbicides, 5 insecticides, and 3 fungicides) commonly used in rice fields in Japan and measured in the present study. Pesticide standards were purchased from Wako Pure Chemical Co. (Japan). Fluoranthene-d10 was purchased from CIL Co. (USA). Naphthalene-d8, anthracene-d10, and chrysene-d12 were purchased from Aldrich Chemical Co. (USA). Florisil, acetone, hexane, and methanol were pesticide reagent-grade (Wako).

Pesticide Residues in Bivalve Organs

37

Fig. 1. Temporal change in pesticide residue concentrations in organs of A. woodiana, C. leana, and river water during the experimental period in 1992. Black bars, white bars, and symbols denote pesticide concentrations in A. woodiana, C. leana, and the river, respectively

Sampling The experiments were conducted from 1 May to 31 July 1992 and from 10 May to 18 August 1994. One week before the field experiments started, C. leana individuals were collected from a stream at the base of Mt. Tsukuba in Ibaraki Prefecture; A. woodiana were purchased from a local pet shop. After acclimation in laboratory stream using ground water, we released 100 individuals of each species into the artificial stream. Before starting the experiments, both species were confirmed to be uncontaminated by pesticides. In the experiment, 10 C. leana and 5 A. woodiana were sampled from the artificial stream once each week. The tissues of the bivalves were dissected into gill (GI), midgut gland (MG), gonad (GO), and the remaining tissues, here called other parts (OP, most of which were

mantle and foot), which were placed in different vessels, weighed, and stored in the dark at ⫺20°C until analysis. Water samples (500 ml) were collected every Monday, Wednesday, and Friday and concentrated by solid phase extraction (C18) on the day of collection.

Analysis Extraction of Pesticide Residues in River Water: The river water samples were filtered through a Whatman GF/C glass filter and passed through a C18-bond Elute column (Varian, USA) at a flow rate of 5 ml min⫺1. The column was centrifuged at 3,000 rpm for 10 min to remove the sample water. Pesticide residues adsorbed on the column were

38

S. Uno et al.

Fig. 1. Continued

eluted with 1.5 ml acetone into a conical centrifuge glass tube and centrifuged at 1,000 rpm for 10 min. This procedure was repeated twice, followed by a third elution with 0.5 ml acetone. The combined extracts were then spiked with naphthalene-d8, anthracene-d10, fluoranthene-d10, and chrysene-d12, or azobenzene and triphenyl phosphate as internal standards. Extraction of Pesticide Residues in Bivalves: The extraction of pesticide residues in the bivalves was carried out by the supercritical fluid extraction (SFE) method using a model SFE-400 (Supelco, USA). An auxiliary pump (Model 576, GL Science, Japan) was used to add a modifier consisting of methanol and water (2:1 v/v). The dynamic flow of supercritical fluid was maintained by a restrictor (20 cm long ⫻ 50 ␮m ID, capillary fused-silica tubing, SGE, Australia). A sample trap was

made by inserting the capillary outlets into a glass vessel containing 1 ml methanol. The operational temperature and pressure of the supercritical mixture containing 10% (v/v) of modifier (methanol) were 50°C and 150 atm, respectively. The flow rate of the liquid CO2 was 1.0 ml min⫺1. Each frozen organ of A. woodiana and C. leana was thawed, shelled, and then freeze-dried. Freeze-dried subsamples (equivalent to about 1 g wet weight) were transferred to an extraction vessel and extracted for 40 min by the SFE method. Three milliliters distilled water was added to the extract, and the aqueous solution was extracted with 3 ml hexane. After shaking and centrifuging at 3,000 rpm for 10 min, the upper phase (hexane) was collected using a glass pipette. This procedure was repeated twice, and the hexane extracts were combined. The combined extract was concentrated to 1 ml under a nitrogen stream. This concentrated extract was loaded on a Pasteur pipette column

Pesticide Residues in Bivalve Organs

39

Fig. 1. Continued

packed with florisil, heated at 450°C for 24 h, washed with 5 ml hexane, and eluted by 5 ml acetone. The eluent was concentrated to 1 ml under a nitrogen stream, and internal standards (naphthalene-d8, anthracene-d10, fluoranthene-d10, and chrysene-d12, or azobenzene and triphenyl phosphate) were added to the concentrate. Gas Chromatographic Analysis: Measurements of the 17 pesticides (9 herbicides, 5 insecticides, and 3 fungicides) commonly used in rice fields (Table 1) were conducted using a gas chromatograph (HP-5890 II; Hewlett Packard, USA) equipped with a mass spectrometer (HP5971A, SIM mode), or an NPD detector with an HP-3395 integrator. The peak area was used for quantification. One microliter of sample was injected by an HP-7673A automatic sampler into a DB-5 capillary column (0.25 mm ID ⫻ 30 m, film thickness 0.25 ␮m; J&W Scientific,

USA) under splitless injection mode. The injector and detector temperatures were 260°C. The oven temperature of the column was incrementally increased as follows: 80°C to 200°C at 10°C min⫺1, 200°C to 220°C at 2°C min⫺1, 220°C to 260°C at 8°C min⫺1, and then maintained at 260°C for 10 min. The detection limits of all pesticides were below 15 ng L⫺1 for water sample and 5 ng g⫺1 (fresh weight) for soft tissue sample, respectively, when 1 ␮l of the extract was injected.

Statistical Method Statistical differences for the changes between A. woodiana and C. corbicula were evaluated using a paired t test, at the p ⫽ 0.05 significance level.

40

S. Uno et al.

Fig. 2. Temporal change in pesticide residue concentrations in organs of A. woodiana, C. leana, and river water during the experimental period in 1994. Bars and symbols are the same as those in Figure 1

Results and Discussion Changes in Pesticide Residue Concentrations in A. woodiana Organs Residues of seven pesticides were detected in A. woodiana in 1992 (molinate, thiobencarb, butachlor, oxadiazon, CNP, IBP, and diazinon) and five in 1994 (molinate, thiobencarb, oxadiazon, IBP, and diazinon) (Figure 1 and 2). Thiobencarb, molinate, and CNP residues in A. woodiana organs were detected in relatively high frequency, but they were quickly eliminated

when concentrations of these pesticide residues decreased in the river. Although oxadiazon, butachlor, IBP, and diazinon residues were detected in the river at high frequency, they were detected only a few times in A. woodiana before starting the experiments. The concentrations of thiobencarb and CNP residues in GO and MG were higher than those in GI and OP. For example, highest concentrations of CNP residues were 930 ng g⫺1 in GO, 640 ng g⫺1 in MG, 390 ng g⫺1 in GI, and 330 ng g⫺1 in OP (22 May 1992, Figure 1C), respectively. Oxadiazon levels followed a similar pattern. However, molinate residue was detected at high concentrations in GI: 140 ng g⫺1 (29 May

Pesticide Residues in Bivalve Organs

41

Fig. 2. Continued

1992, Figure 1A) and 290 ng g⫺1 (24 May 1994, Figure 2A), respectively. Except for GI, molinate also accumulated in GO. The pattern of changes in concentrations of molinate and thiobencarb residues in GI was clearly different from that in MG, GO, and OP. Increases in the concentrations of molinate and thiobencarb in GI did not parallel changes in their concentration in the river for 2 to 3 weeks from the beginning of detection. While their concentrations in the river and all organs except GI increased for 2 to 3 weeks (Figure 1A, 1B, 2A, 2B), the concentrations in GI started to decrease. This phenomenon was observed in both years. Bivalves usually adsorb pesticides through the gill and distribute them via the blood into each organ, as do fish. In both, the gill is the first organ exposed to pesticide residues. The changes in

concentrations of molinate and thiobencarb in bivalve gills might be caused by either decreased ventilation for respiration due to gill damage or a self-defense mechanism that avoids adsorption of these toxic pesticide residues. For about 1 month from the middle of May in 1994, high mortality of A. woodiana was observed in the artificial stream (Figure 3), while most C. leana survived. The highest concentrations of molinate and thiobencarb residues in the river were observed 5–7 days before the maximum mortality rate of the bivalves. These results suggest that although the accumulation concentrations and remaining period of these pesticide residues in A. woodiana were lower and shorter, respectively, than those in C. leana, the toxicity of these pesticides to A. woodiana was extremely high.

42

Fig. 3. Changes in individual deaths of A. woodiana and in the concentration of molinate and thiobencarb in river water (Kokai River, 1994). Bars denote the individual deaths of A. woodiana; triangles and circles represent the concentrations of molinate and thiobencarb, respectively, in the river

Changes in Pesticide Residue Concentrations in C. leana Organs Figure 1 and 2 also show the changes in concentration of residues of several pesticide residues in C. leana organs in 1992 and 1994. Molinate, mefenacet, butachlor, and pretilachlor were quickly eliminated from all organs, although these pesticides were still detectable in the river. However, thiobencarb and CNP accumulated in the organs at high levels for 1–3 months. In particular, the concentration of thiobencarb residue in all organs remained high until the end of the experiment; the highest concentrations were 15.70 ␮g g⫺1 (19 June 1992, Figure 1B) and 12.45 ␮g g⫺1 (22 June 1994, Figure 2B), respectively, for MG, and 15.80 ␮g g⫺1 (26 June 1992) and 16.40 ␮g g⫺1 (22 June 1994), respectively, for GO. All pesticide residues detected; CNP, oxadiazon, molinate, mefenacet, butachlor, and pretilachlor as well as thiobencarb were accumulated at high levels in MG and GO.

Interspecies Differences in the Concentrations of Accumulated Pesticide Residues in Organs of A. woodiana and C. leana A. woodiana and C. leana are known to have similar respiratory systems and feeding habits, but interspecies differences in concentrations and remaining period of pesticide residues in each organ were clearly observed. For example, the highest concentrations of thiobencarb residue in MG and GO of C. leana were 35 and 40 times, respectively, higher than those of A. woodiana. As the result of statistical evaluation, the differences for the changes between two species was significant (p ⬍ 0.05), except for molinate in GI and OP. Because the residue period of molinate was short in two bivalves and this tendency

S. Uno et al.

was particularly clear in GI and OP, the significance differences were not shown. Molinate, thiobencarb, and CNP were always detected in all organs of A. woodiana during the experimental periods, but their concentrations were only few times higher than those after early July when the pesticide application period was over. Molinate, thiobencarb, oxadiazon, and CNP were always detected in all organs of C. leana even after late July. In Japan, the use of CNP was banned in 1994 because it was suspected of inducing gallbladder cancer in humans. However, CNP was detected in all organs of C. leana for 6 weeks in 1994 (13– 470 ng/g), although the pesticide was undetectable in river water and in A. woodiana. These results may suggest that CNP residue remained in the river sediment, from which C. leana ingested it in floating particles resuspended from the river bed. This would suggest that the filtering and bioaccumulative characteristics (both of bioconcentration and biomagnification) of the two species may be different. In general, MG and GO in both species accumulated more pesticide residues than did GI and OP. These tendencies were similar to those in other shellfish. Roberts (1975) exposed M. edulis and C. opercularis to endosulfan for 36 days under laboratory conditions and found that its order of concentration in M. edulis was midgut gland ⬎ gill ⬎ postal ligament ⬎ mantle and reproductive organ, whereas that in C. opercularis was midgut gland ⬎ reproductive organ ⬎ mantle ⬎ ligament ⬎ gill. Watanabe et al. (1985) reported that M. edulis exposed to molinate, thiobencarb, CNP, and chromethoxinyl under laboratory conditions accumulated the pesticide residues at higher levels in gonad and midgut gland, which are much higher in lipid content than are the gill and other organs. In fish, Gingerich (1986) reported that [14C]rotenone was accumulated in the order of hepatobiliary system, bile, intestine, heart, lateral line swimming muscle, and posterior kidney of rainbow trout under laboratory conditions. Gunkel and Streit (1980) reported that [14C]ring-labeled atrazine was rapidly accumulated in liver, brain, gill, intestine, and gallbladder of Coregonus fera in laboratory experiments and concluded that high accumulation rates occur in organs of fish that have high blood circulation (e.g., liver, brain, gill, intestine). Since the blood circulation of bivalves is an open blood vascular system (in contrast to the closed circulatory system of fish), there should be significant differences in the accumulative processes between bivalves and fish. All pesticide residues in each organ of A. woodiana were under detection limits after they disappeared in river water. Furthermore, the changes of concentration for almost pesticides between in each organ of A. woodiana and river water were similar. These results suggest the intake and elimination rate of pesticides in each organ of A. woodiana were strongly influenced by their concentration in river water. On the other hand, thiobencarb, CNP, and oxadiazon in C. leana remained at high levels and their elimination rate would be low because they were detected even after they become undetectable in the river water (Table 2). Therefore, the elimination ability of pesticides in C. leana could be inferior to that of A. woodiana. Because the lipophilicities of CNP and oxadiazon are high relatively (Table 1), the elimination rates of these pesticides in C. leana could be slow. But that of thiobencarb is lower than those in butachlor and pretilachlor. Butachlor and pretilachlor were detected frequently in experiment periods, but were undetectable immediately and disappeared in river water. There-

Pesticide Residues in Bivalve Organs

43

Table 2. The elimination rate constants and half lives of pesticides in each organ for Corbicula leana 1992

Gill Midgut gland Gonad Other a b

1994

Thiobencarb

CNP

Oxadiazone

Thiobencarb

CNP

Oxadiazone

a

0.061 (11.6) 0.081 (8.5) 0.087 (7.9) 0.091 (7.6)

0.087 (8.0) 0.074 (9.4) 0.053 (13.2) 0.11 (6.4)

0.018 (38.5) 0.015 (46.2) 0.033 (21.0) 0.019 (36.5)

0.35 (2.0) 0.074 (9.4) 0.086 (5.1) 0.035 (19.8)

0.075 (9.2) 0.072 (9.6) 0.055 (12.6) 0.036 (19.5)

0.063 0.054 0.043 0.052

b

(11.1) (12.9) (16.2) (13.4)

Elimination rate constant (day⫺1) Half-lives in C. leana (day)

Fig. 4. Temporal changes in the percentages of pesticide residues in each organ to whole soft tissue (A. woodiana, 1992). The vertical hatching denotes the gill; blank area, gonad; horizontal hatching, midgut gland; oblique hatching, other parts; and black circles, whole soft tissue

fore, we could not find clear relations between the lipophilicities of pesticides and the elimination rates. To examine the accumulative characteristics of three pesticides in the main organs of the bivalves, the proportion of

pesticide in each organ relative to that in whole soft tissue in A. woodiana was calculated as a percentage (Figure 4 and 5), using the concentrations of pesticide residues in whole soft tissue estimated from those in each organ and the organ

44

S. Uno et al.

Fig. 5. Temporal change in the percentage of each pesticide in each organ to whole soft tissue (A. woodiana, 1994). Symbols and patterns are the same as those in Figure 4

weights. The results indicate that GI, GO, and MG in A. woodiana accumulated more than 60% of the residues. The percentages of thiobencarb and molinate residues in GI decreased with time, although their concentrations in whole soft tissue increased. This may mean that the increase occurred due to biomagnification, whereas self-defense mechanisms in the gill worked. However, the percentages of thiobencarb and molinate in GO increased with time while their concentrations in whole soft tissue were decreasing. This result may imply that accumulated pesticide residues in GI were distributed normally into each organ, resulting in their reduced concentrations in GI, and further accumulation did not occur through the gill. The percentages of CNP residue in A. woodiana organs were almost constant (mean: GI, 18%; MG, 39%; GO, 30%; and OP, 13%, respectively). These results may imply that CNP residue was distributed rapidly into each organ, was eliminated from each organ at a similar rate, and did not remain in any specific organ. Molinate, thiobencarb, and CNP in A. woodiana were more rapidly eliminated from OP than from GI, MG, and GO, while these pesticide residues remained in GO. The percentages of thiobencarb in each organ in C. leana did not vary greatly: 6% (1992 and 1994) for GI; 21% (1992) and 25% (1994) for MG; 26% (1992) and 27% (1994) for GO; and 46% (1992) and 43% (1994) for OP, respectively (Figures 6 and 7). These results imply that after thiobencarb was adsorbed into the body through the gill, it was immediately distributed into each organ and eliminated at a similar rate to its uptake. Oxadiazon and mefenacet showed the same tendencies. The concentra-

tion of thiobencarb residue was still high in each organ at the end of the experiment, implying that it can remain in all organs. However, CNP residue remained mostly in MG and GO. Molinate had the same tendency. The variations in CNP content in 1994, when the use of CNP was banned, were different from those in 1992. It was likely that, in 1992, C. leana accumulated CNP due to bioconcentration, but in 1994 the accumulation occurred due to biomagnification. The patterns of variation in concentrations of pesticide residues in the organs of both bivalves were almost the same in the two sampled years. Bivalves control the volume of water pumped into the pallial cavity and the volume of water filtered can vary from 0 to 4 L h⫺1 (Rhodes et al. 1997). These differences can result in the different accumulation concentrations. Some pesticides accumulated in high concentrations in C. leana, but in A. woodiana the extremely high in concentrations only in GI were not observed. All organs of C. leana, except for GI, accumulated pesticide residues at much higher levels than did those in A. woodiana, and some remained in the C. leana organs for a long period. However, most C. leana survived when high mortalities of A. woodiana were observed. Hatakeyama (1989) reported that reproduction in the guppy Poecilia reticulata exposed to more than 5 ␮g L⫺1 CNP in the laboratory was significantly depressed compared with that in control. In our experiment in 1992, the highest CNP residue concentration in the river water was 0.27 ␮g L⫺1, but CNP in GO had been detected at ppm levels for 6 weeks. Since elimination rates in bivalves are much lower than those in fish (Esser 1986; Stegeman and Lech 1991; Rhodes et al. 1997), the

Pesticide Residues in Bivalve Organs

45

Fig. 6. Temporal change in the percentage of each pesticide in each organ to whole soft tissue (C. leana, 1992). Symbols and patterns are the same as in Figure 4

presence of CNP at ppt levels would affect reproduction of C. leana in the field.

Conclusions In the present study, the accumulative characteristics of pesticide residues in the organs of two bivalves, C. leana and A. woodiana, were examined using a small artificial stream under natural conditions. The unusual changes in concentration of molinate and thiobencarb residues in GI of

A. woodiana suggested that this organ might be damaged by the two pesticides. The midgut gland and gonad of both bivalves were accumulative organs of pesticide residues in the river water. Interspecies differences appeared in the high concentrations and clearance times of pesticide residues in the organs. The difference in distribution patterns of pesticide residues in the organs might be attributed to two factors; first, they are immediately distributed into each organ after intake and eliminated at similar rates to intake rates; second, they can remain at high levels in the gonad and midgut gland for a long period.

46

S. Uno et al.

Fig. 7. Temporal change in the percentage of each pesticide in each organ to whole soft tissue (C. leana, 1994). Symbols and patterns are the same as those in Figure 4

Acknowledgments. The authors wish to thank S. Sasaki and K. Kawabe for their assistance.

References Barnes RD (1968) Invertebrate zoology. WB Saunders Company, Philadelphia, PA Connell DW (1995) Prediction of bioconcentration and related lethal and sublethal effects with aquatic organisms. Mar Pollut Bull 31:201–205 Esser HO (1986) A review of the correlation between physicochemical properties and bioaccumulation. Pestic Sci 17:265–276 Gingerich WH (1986) Tissue distribution and elimination of rotenone in rainbow trout. Aquat Toxicol 8:27– 40

Gunkel G, Streit B (1980) Mechanisms of bioaccumulation of a herbicide (atrazine, s-triazine) in a freshwater mollusk (Ancylus fluviatilis Mull) and a fish (Coregonus fera Jurine). Water Res 14:1573–1584 Hatakeyama S (1989) Effect of a herbicide, chlornitrofen (2,3,6trichloropehnyl ether), on the growth and reproduction of the guppy (Poecilia reticulata) through water and food. Aquat Toxicol 15:181–196 Hatakeyama S, Shiraishi H, Sugaya Y (1991) Monitoring of the overall pesticide toxicity of river water to aquatic organisms using a freshwater shrimp, Paratya compressa improvisa. Chemosphere 22:229 –235 Roberts D (1975) Differential uptake of endosulfan by the tissues of Mytilus edulis. Bull Environ Contam Toxicol 13:170 –176 Rhodes LD, Gardner GR, Van Beneden RJ (1997) Short-term tissue

Pesticide Residues in Bivalve Organs

distribution, depuration and possible gene expression effects of [3H]TCDD exposure in soft-shell clams (Mya arenaria). Environ Toxicol Chem 16:1888 –1894 Shiino S (1969) Biology of invertebrates. Baifukan, Tokyo Stegeman JJ, Lech JJ (1991) Cytochrome P-450 monooxygenase systems in aquatic species: carcinogen metabolism and biomarkers for carcinogen and pollutant exposure. Environ Health Perspect 90:101–109 Uno S, Shiraishi H, Hatakeyama S, Otsuki A (1997) Uptake and

47

depuration kinetics and BCFs of several pesticides in three species of shellfish (Corbicula leana, Corbicula japonica, and Cipangopludina chinensis): comparison between field and laboratory experiment. Aquat Toxicol 39:23– 43 Watanabe S, Watanabe S, Ito K (1985) Accumulation and excretion of herbicides in various tissues of mussel. J Food Hyg Soc 26:496 – 499 Yamagishi T, Akiyama K (1981) 1,3,5-Trichloro-2-(4-nitrophnoxy) benzene in fish, shellfish and seawater in Tokyo Bay, 1977–1979. Arch Environ Contam Toxicol 10:627– 635